Gene regulatory networks establish the body plans of complex multicellular animals. The main components of these networks are transcription factors and cis-regulatory elements (CREs) that contain unique combinations of transcription factor binding sites. Transgenic experiments have shown that CREs control spatial and temporal patterns of gene expression, but how binding events at the CRE level are converted into these patterns is still poorly understood. In humans, mutations that disrupt transcription factor activit or CRE DNA sequences can cause birth defects and disease states, including cancer. Here we propose to study CRE- mediated embryo patterning in the early Drosophila embryo, with a specific focus on CREs that respond to the maternal transcription factor Bicoid (Bcd). In previous work, we identified 66 Bcd-dependent CREs, which were validated by reporter genes in transgenic embryos. All 66 are directly activated by Bcd, but each contains a unique set of transcription factor-binding sites, and drives an expression pattern that appears as a band(s) or stripe(s) at a specific position(s) along the anterior-posterior (AP) axis. Expression pattern boundaries represent positions where genes make on/off decisions, and foreshadow boundaries between cells of different fates later in development. The work proposed here will focus on two major goals. First, we want to perform a deep, quantitative analysis of transcriptional activation by a single model CRE (hunchback P2E) that is activated by BCD. Two specific questions will be addressed: 1. how do individual binding sites, groups of binding sites, and combinations of sites for different factors contribute to transcriptional activation? We will combine DNA-binding data of different types to better define functional motifs, extensively mutate the CRE, and assay three specific output parameters using live imaging in close collaboration with Thomas Gregor's lab. 2. How does this Bcd-dependent CRE choose its specific basal promoter? We will use a split reporter system and single molecule FISH to identify basal promoter sequences that allow or prevent activation by Bcd, and CRISPR/Cas9 gene editing to test the importance of promoter choice in vivo. The second major goal will be to gain a conceptually complete understanding of the regulatory network that creates Bcd-dependent gene expression patterns. We have identified three proteins that function in broad activation, and nine candidate repressors expressed in discrete domains that together cover the entire embryonic region where activation can occur. We will use reporter gene manipulations and single molecule FISH to test how repressor gradients shape boundaries of gene expression, and genetics and reporter genes to test the roles of several newly identified repressors that prevent activation in anterior regions. Our hypothesis is that the expression patterns driven by all Bcd-dependent CREs are computed by specific combinations of binding sites for these twelve factors. These experiments will make significant contributions to our general understanding of how CREs functions in activation and repression events, and how embryos establish body plans.